Several methods have been developed to detect protein-protein interactions, all with their advantages and limitations. Co-purification of proteins and co-immunoprecipitation were amongst the first techniques used. However, these methods are tedious and do not allow high throughput screening. Moreover, they require lysis corrupting the normal cellular context. A major breakthrough was obtained by the introduction of the genetic approaches, of which the yeast two-hybrid (Fields and Song, 1989) is the most important one. Although this technique became widely used, it has several drawbacks. The fusion proteins need to be translocated to the nucleus, which is not always evident. Proteins with intrinsic transcription activation properties may cause false positives. Moreover, interactions that are dependent upon secondary modifications of the protein such as phosphorylation cannot be easily detected.
Several alternative systems have been developed to solve one or more of these problems.
Approaches based on phage display do avoid the nuclear translocation. WO9002809 describes how a binding protein can be displayed on the surface of a genetic package, such as a filamentous phage, whereby the gene encoding the binding protein is packaged inside the phage. Phages, which bear the binding protein that recognizes the target molecule, are isolated and amplified. Several improvements of the phage display approach have been proposed, as described, e.g., in WO9220791, WO9710330 and WO9732017.
However, all these methods suffer from the difficulties that are inherent at the phage display methodology: the proteins need to be exposed at the phage surface and are so exposed to an environment that is not physiological relevant for the in vivo interaction. Moreover, when screening a phage library, there will be a competition between the phages that results in a selection of the high affinity binders.
U.S. Pat. No. 5,637,463 describes an improvement of the yeast two-hybrid system, whereby can be screened for modification dependent protein-protein interactions. However, this method relies on the co-expression of the modifying enzyme, which will exert its activity in the cytoplasm and may modify other enzymes than the one involved in the protein-protein interaction, which may on its turn affect the viability of the host organism.
An interesting evolution is described in U.S. Pat. No. 5,776,689, by the so-called protein recruitment system. Protein-protein interactions are detected by recruitment of a guanine nucleotide exchange factor (Sos) to the plasma membrane, where Sos activates a Ras reporter molecule. This results in the survival of the cell that otherwise would not survive in the culture conditions used. Although this method has certainly the advantage that the protein-protein interaction takes place under physiological conditions in the submembranary space, it has several drawbacks. Modification-dependent interactions cannot be detected. Moreover, the method is using the pleiotropic Ras pathway, which may cause technical complications. Most of these drawbacks were solved by the Mammalian Protein-Protein Interaction Trap (MAPPIT) described in WO0190188, using recruitment of a prey to a cytokine type of receptor, fused to a bait. However, although this method allows to study protein-protein interactions under physiological conditions, it is not suitable to study interactions involving integral membrane proteins, particularly multispan membrane proteins, including GPCR's.
Methods for studying the interaction of proteins with a GPCR are mainly focused on ligand-receptor binding. WO9834948 discloses a GPCR wherein the amino terminus is replaced by the amino-terminus of a self-activating receptor, and the use of this construct for the detection of agonists and antagonists. WO2004099419 discloses a ligand upregulatable GPCR, and the use of this construct to screen ligands. WO0158923 describes methods for detecting GPCR activity, methods for assaying GPCR activity and methods for screening GPCR ligands, G-protein-coupled receptor kinase activity and compounds that interact with the GPCR regulatory process, by an enzyme complementation assay. However, this system is rather insensitive, with a maximal window of a factor 2 at the highest concentrations of agonist or antagonist used. Moreover, the system needs a mutation in arrestin, to improve arrestin binding, in order to obtain the required sensitivity.